The overall purpose of this project is to identify activities produced by marrow stromal cells that can stimulate human stem cell (SC) proliferation without compromising their long-term repopulating ability. Achieving this goal would allow for ex vivo expansion of SC, increase the efficiency of gene transfer into SC, and facilitate their engraftment. This in turn would increase the usefulness of SC transplantation for the treatment of genetic and acquired diseases. The proposed studies are based on the premise that SC with self- replicating potential are quiescent and can be distinguished from more committed progenitor cells (PC) by differential gene expression. The reduced levels of cytokine receptor mRNA and the absence of a cytokine response observed in SC-enriched populations support this premise. We propose to exploit these differences in two ways. First, we propose to use two short-term assays of cell proliferation to screen for activities that simulate proliferation of SC-enriched cells. One assay measures DNA content of cells exposed to soluble cytokines or stromal cell supernatants; the other uses in situ staining and confocal microscopy to measure changes in number and viability of cells cultured in contact with stromal cells. Both provide a very quick readout of cellular proliferation, which is needed to screen for stimulating activities. The second component of the proposed studies is to determine if the stimulated SC population has retained its SC potential. Initially this will require quantitating the number of long-term culture-initiating cells (LTC-IC) before and after stimulation. This assay, although considered to be the best in vitro approximation of SC function, takes 7 weeks. We propose to develop a more rapid assay by defining gene expression that will distinguish between SC and PC, and once defined, use quantitative RT- PCR of the relevant mRNA to determine how enriched starting populations are for SC versus PC, and whether stromal-derived activities alter this. The final determination of SC function will involve transfecting the isolated population of cells with a retrovirus and using the marked cells for autologous rescue after marrow ablation. Stromal cells that will be tested for SC-stimulating activity have been immortalized with HPV16 E6/E7 genes, which degrade the negative cell cycle regulators p53 and rb. Eleven immortalized lines, AT-1 - AT-11, have been partially characterized, and one, designated AT-5, supports proliferation of isolated CD34+ cells in the absence of serum or exogenous cytokines. These preliminary studies on CD34+ cells confirm the utility of the short- term proliferation assays. Studies must now be performed on more defined subsets of CD34+ cells. Once a stromal activity is identified, standard biochemical and molecular approaches will be used to characterize the activity and isolate the gene that encodes it.